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KMID : 1023520140370040263
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Korean Journal of Veterinary Service
2014 Volume.37 No. 4 p.263 ~ p.271
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One step multiplex RT-PCR preventing DNA carryover contamination for differential diagnosis of swine influenza viruses
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Kim Hee-Jung
Kim Eun-Mi
Yeo Sang-Geon
Park Choi-Kyu
Shin Yeun-Kyung
Song Jae-Young
Kim Sung-Hee
Lee Kyoung-Ki
Lee Myoung-Heon
Kim Young-Hwa
Park Jun-Cheol
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Abstract
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In this study, we developed a cost and time saving one-step multiplex RT-PCR for the simultaneous detection and differentiation of swine influenza viruses (SIV) and 2009 pandemic influenza H1N1 virus (pH1N1). The one-step multiplex RT-PCR using four sets of primer was confirmed to be capable of detection of all SIV subtypes and differential diagnosis of major SIV subtype H1, H3 and pH1N1 on individual or mixed viral culture samples. The sensitivity of the multiplex RT-PCR was determined to be at least 2-6 HA/25 μL of the presented SIVs, providing sufficient efficacy for a routine SIV monitoring in diagnostic laboratories. In addition, compared with the conventional RT-PCR methods that cannot avoid the carryover DNA contamination, the developed RT-PCR applied with the uracil DNA glycosylase (UNG) system was proven to prevent a false positive reaction by carryover contamination of the pre-amplified DNA. In conclusion, the one-step RT-PCR with UNG system could be applicable to detect and differentiate of SIV from the viral cultures without worry of carryover DNA contamination in clinical laboratories.
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KEYWORD
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Swine influenza viruse (SIV), Multiplex RT-PCR, Uracil DNA glycosylase (UNG), Differential diagnosis
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